ION CHANNELS, RECEPTORS AND TRANSPORTERS

Sodium/calcium exchanger is upregulated by sulfide signaling,forms complex with the β1 and β3 but not β2 adrenergicreceptors, and induces apoptosis

Jana Markova & Sona Hudecova & Andrea Soltysova &

Marta Sirova & Lucia Csaderova & Lubomira Lencesova &

Karol Ondrias & Olga Krizanova

Received: 18 July 2013 /Revised: 6 September 2013 /Accepted: 20 September 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract Hydrogen sulfide (H2S) as a novel gasotransmitterregulates variety of processes, including calcium transportsystems. Sodium calcium exchanger (NCX) is one of thekey players in a regulation calcium homeostasis. Thus, theaims of our work were to determine effect of sulfide signalingon the NCX type 1 (NCX1) expression and function in HeLacells, to investigate the relationship of β-adrenergic receptorswith the NCX1 in the presence and/or absence of H2S, and todetermine physiological importance of this potential commu-nication. As a H2S donor, we used morpholin-4-ium-4-methoxyphenyl(morpholino) phosphinodithioate—GYY4137.We observed increased levels of the NCX1 mRNA, protein,and activity after 24 h of GYY4137 treatment. This increasewas accompanied by elevated cAMP due to the GYY4137treatment, which was completely abolished, when NCX1 wassilenced. Increased cAMP levels would point to upregulation ofβ-adrenergic receptors. Indeed, GYY4137 increased expres-sion of β1 and β3 (but not β2) adrenergic receptors. Thesereceptors co-precipitated, co-localized with the NCX1, andinduced apoptosis in the presence of H2S. Our results suggestthat sulfide signaling plays a role in regulation of the NCX1,β1and β3 adrenergic receptors, their co-localization, and stimula-tion of apoptosis, which might be of a potential importance incancer treatment.

Keywords Hydrogen sulfide . Sodium-calcium exchanger .

β-Adrenergic receptors . Apoptosis . Cancer cells

Introduction

Sodium calcium exchanger (NCX) is one of the key players inthe regulation of an intracellular calcium homeostasis. Threetypes of the NCX were found and characterized up to now;however, type 1 is the best studied one. Although majority ofpapers dealing with the NCX1 comes from the cardiac tissue[3, 18, 22], importance of this calcium transporter was docu-mented also in other types of cells and tissues, e.g. brain [28,59], endocrine system and in immune system [55]. Calciuminflux through the reverse mode NCX is required for activa-tion and targeting of the PKCα to the plasma membrane as anessential step for vascular endothelial growth factor (VEGF)-induced ERK1/2 phosphorylation and downstream EC func-tions in angiogenesis [2]. NCX can play a role also in theapoptosis development [8, 41]. Cuozzo and coworkers [8]have shown that the cardiac glycoside ouabain is cytotoxicfor the lymphoma-derived cell line U937 and it can activate asurvival pathway, in which NCX and p38 MAPK are in-volved. Calcium influx via the reverse NCX is involved inthe cascade of NO-induced neuronal apoptosis and NO acti-vation of the NCX through the guanylate cyclase/PKG path-way [41]. NCX overexpression also induces apoptosisthrough the endoplasmic reticulum stress [10].

Signaling through the individual types of adrenergic recep-tors (AR) was described only in cardiac tissue and belongs tothe most important regulations of the NCX. NCX1 is upreg-ulated at both transcriptional and protein levels with the β-ARstimulation in neonatal rat cardiomyocytes and in the adult ratheart [16, 17]. Mani and coworkers [36] have demonstratedthat majority of the β-AR-stimulated NCX1 upregulation is

J. Markova : S. Hudecova :A. Soltysova :M. Sirova :L. Lencesova :K. Ondrias :O. Krizanova (*)Institute of Molecular Physiology and Genetics, Slovak Academy ofSciences (SAS), Vlarska 5, 833 34 Bratislava, Slovak Republice-mail: olga.krizanova@savba.sk

L. CsaderovaInstitute of Virology, SAS, Bratislava, Slovakia

A. SoltysovaDepartment of Molecular Biology, Faculty of Natural Sciences,Comenius University, Bratislava, Slovakia

Pflugers Arch - Eur J PhysiolDOI 10.1007/s00424-013-1366-1

mediated by a Ca2+/calmodulin kinase II dependent pathwayresulting in the ordered recruitment of JunB, followed by c-Jun homodimers, to the proximal Ncx1 AP-1 elements.Results from our recent work suggested that isoproterenolfurther enhanced NCX1 expression in apoptotic cells throughthe development of endoplasmic reticulum stress [23].

β-Adrenergic receptors transduce signals from the cate-cholamines—noradrenaline and adrenaline—to Gs protein,which in turn activates its effector adenylyl cyclase to producethe second messenger cAMP. The cAMP further activates itsmajor cellular effector cAMP-dependent protein kinase(PKA). Physiological (and pathophysiological) relevance ofthese signaling pathways is described predominantly in a heart[14, 54, 56], but also in other processes, like bone remodeling[11], aging [50], and in the development of apoptosis [19, 27].During induction of apoptosis, all three types of β-ARs wereupregulated [31].

H2S is a novel gasotransmitter—endogenous gas with theimportant physiological function [60]. Although originallyconsidered as an organic waste, H2S was found as an endog-enous gas in a variety of tissues. It has been shown thatendogenously generated H2S is oxidized to sulfate, or storedin proteins, where it may be released to cause a physiologicalstimulus [26]. Free H2S concentration in the blood and also intissues is in the range of 10–20 nmol/l [15, 26]. However, it isassumed that in intracellular locations where H2S synthesizingenzymes are highly concentrated, the free H2S might tempo-rarily become highly concentrated in the microenvironmentbefore it has time to diffuse away, be bound, or be oxidized[33]. Taking into account the H2S toxicity, it was hypothesizedthat physiological H2S concentrations in microenvironmentsdo not exceed 100 μmol/l [33]. Nevertheless, it was alreadyshown that also exogenous H2S might be of a potential ther-apeutic benefit in several diseases, e.g., cardiovascular, onco-logic, and immunological [47, 61]. Breakthrough in the effortin linking the H2S level and functional changes came whenAbe and Kimura [1] reported that fast H2S donor, NaHS,facilitated the induction of hippocampal long-term potentia-tion at micromolar concentrations. Nowadays, several targetsof the H2S signaling are known [46, 61] including ion channelsand transporters [13, 29, 40, 68]. One of the metabolic featuresof the H2S described is an increase in the cytosolic calcium.Recently, we have shown that H2S is involved in both inositol1,4,5-trisphosphate-induced calcium signaling and inductionof apoptosis, possibly through the activation of ER stress [30].

Nevertheless, both pro- [5, 12, 25, 62] and anti-apoptoticeffects [21, 64, 67] of the H2S have been reported up to now.Differences might account for different cell types and differentlevels of the H2S.

Goal of the present study was to elucidate the effect of slowsulfide donor—GYY4137—on the NCX1 expression andactivity in HeLa cells and to determine the possible impacton apoptosis induction. Because of shown β-AR impact on

the cardiac expression of the NCX [17], interaction with theindividual types of β-ARs was studied as well.

Material and methods

Cell cultivation and treatment

A human cervix carcinoma cell line (HeLa; German Collectionof Microorganisms and Cell Cultures, Braunschweig,Germany) was cultured in Minimal Essential Medium ofDulbecco (DMEM; Sigma, USA) with high glucose (4.5 g/l)and L-glutamine (300 μg/ml), supplemented with 10 % fetalbovine serum (Sigma, USA), penicillin (Calbiochem, USA;100 U/ml) and streptomycin (Calbiochem, USA; 100 μg/ml).Cells were cultured in a water-saturated atmosphere at 37 °Cand 5 % CO2. For experiments, HeLa cells were grown in 6-well plates or 25 ml flasks to the confluence max 70 %. Cellswere treated for 24 h with slowH2S release donor morpholin-4-ium-4-methoxyphenyl (morpholino) phosphinodithioate(GYY4137; Cayman Chemical, USA; 10 μM). Concentrationof the GYY4137 was chosen based on the experiments pub-lished previously [30]. In order to check involvement of theadrenergic stimulation on the NCX, some group of cells weretreated for 24 hwith nonspecific blocker ofβ1 andβ2 receptorspropranolol hydrochloride (Calbiochem, USA; 10 μM), specif-ic β1 blocker CGP-20712A methanesulfonate salt (Sigma-Aldrich, USA; 10 μM), specific β2 blocker ICI 118,551 hy-drochloride (Tocris Bioscience, UK; 10 μM) and specific β3blocker SR 59230 A (Tocris Bioscience, UK; 10 μM). To showthe involvement of GYY4137, dithiothreitol (DTT; Fluka,Switzerland, 10 μM) was used.

RNA preparation and relative quantification of mRNA levelsby RT-PCR

Population of total RNAs was isolated by TRI Reagent(Sigma, USA). Briefly, cells were scraped and homogenizedby pipette tip in sterile water, and afterwards, TRI Reagentwas added. After 5 min, the homogenate was extracted bychloroform. RNAs in the aqueous phase were precipitated byisopropanol. RNA pellet was washed with 75 % ethanol andstored in 96 % ethanol at −70 °C. The purity and integrity ofisolated RNAs were checked on GeneQuant Pro spectropho-tometer (Amersham Biosciences, UK). Reverse transcriptionwas performed using 1.5 μg of total RNAs and Ready-To-GoRT-PCR Beads (GE Healthcare-Life Sciences, UK) withpd(N6) primer. PCR specific for the NCX1 was carried outafterwards using primers NCXLA sense: 5′-TCC CAT CTGTGT TGT GTT CGC-3′ and NCXLB antisense: 5′-TCATCTTGG TCC CTC TCA TC-3′ (gene ID: 6453726), for Beta1using primers Beta1fw sense: 5′-GGT GGC CCC GAA CGGCACCACCAGCA-3′ and Beta1rev antisense: 5′-CGAGCC

Pflugers Arch - Eur J Physiol

GCT GTC TCA GCA GTG GAC A-3′ (gene ID: 1781999),for Beta2 using primers Beta2fw sense: 5′-CTG GTC ATGGGC CTG GCA GT-3′ and Beta2rev antisense: 5′-CAT GATGATGCC TAA CGTCT-3′ (gene ID: 178201), and for Beta3using primers Beta3fw sense: 5′-GCA ACA ACT CTG TTGATC AG-3′ and Beta3rev antisense: 5′-ATC TCT CAG ACAGAG AGC AA-3′ (gene ID: 68248547). For AdCy7 primers,AdCy7 1A: 5′-AAC CCG CTA TCT GGA GTC CT-3′ andAdCy7 1B: 5′-TGG GGG ATG CAC TCC TAT CA-3′ (geneID: 228480245) were used. β-Actin and cyclophilin A wereused as a housekeeper gene control for semi-quantitativeevaluation of PCR. For the β-actin, following primers wereused: BA1 sense: 5′-AGG CCG GCT TCG CGGGCGAC-3′and BA2 antisense: 5′-CTC GGG AGC CAC ACG CAGCTC-3′ (gene ID: 28251). For cyclophilin A, primers Cyclofw sense: 5′-CGT GCT CTG AGC ACT GGG GAG AAA-3′and Cyclo rev antisense: 5′-CAT GCC TTC TTT CAC CTTCCC AAA GAC-3′ (gene ID: 114520617) were used. PCRspecific for NCX1 started by initial denaturation at 94 °C andwas followed by 27 cycles of denaturation at 94 °C for 1 min,annealing at 58 °C for 1 min and polymerization at 72 °C for1 min PCR specific for Beta1 started with initial denaturationat 94 °C and was followed by 35 cycles of denaturation at94 °C for 1 min, annealing at 60 °C for 1 min and polymer-ization at 72 °C for 1 min. PCR specific for Beta2 started withinitial denaturation at 94 °C and was followed by 35 cycles ofdenaturation at 94 °C for 1 min, annealing at 61 °C for 1 min,and polymerization at 72 °C for 1 min. PCR specific for Beta3started with initial denaturation at 94 °C and was followed by31 cycles of denaturation at 94 °C for 1 min, annealing at60 °C for 1 min, and polymerization at 72 °C for 1 min. PCRspecific for AdCy7 started with initial denaturation at 94 °Cand was followed by 35 cycles of denaturation at 94 °C for1 min, annealing at 60 °C for 1 min and polymerization at72 °C for 1 min. PCR specific for β-actin and cyclophilin Astarted with initial denaturation at 94 °C and was followed by25 cycles of denaturation at 94 °C for 1 min, annealing at60 °C for 1 min, and polymerization at 72 °C for 1 min. AllPCRs were terminated by final polymerization at 72 °C for7 min. All PCR products were analyzed on 2 % agarose gels.Intensity of individual bands was evaluated by measuring theoptical density per square millimeter and compared relativelyto the β-actin/cyclophilin A on PCBAS 2.0 software.

Real-Time PCR

PCR amplification and detection was performed on thePikoReal 96 Real-Time PCR System (Thermo FisherScientific, Hampshire, UK). SYBR Green Master Mix withROX reference dye (Fermentas, Germany), primers at 5 pmoland 10 % of the reverse transcription product were mixed to afinal volume of 20 μl. Master Mix with primers and templatewere separately loaded onto 96-well plates (Thermo Fisher

Scientific, Hampshire, UK). Plates were centrifuged to removeany air bubbles in the wells. Each sample was run in duplicateand with one “no template” control. Each cycle consisted of15 s at 95 °C, 30 s at 60 °C, and 30 s at 72 °C for 40 cycles afteran initial activating step for 2 min at 50 °C and denaturing stepfor 10 min at 95 °C. Primers used for specific genes were thesame as for the RT-PCR. To monitor the presence ofnonspecific products, routine melting curve analyses wereperformed after the end of amplification. This was done byhigh-resolution data collection during an incremental tempera-ture increase from 60 to 95 °C. Data were analyzed withPikoReal software version 2.1 (Thermo Fisher Scientific,Hampshire, UK) and inspected to determine artifacts (loadingerrors, threshold errors). Baseline levels for each gene werecomputed automatically. Results were analyzed as a peak areafor everywell and quantified relatively fromCq values accordingto the formula ΔΔCq=ΔCqsample −ΔCqhousekeeper, wherethe human cyclophilin A or β-actin was used as housekeeper.

Gene silencing

For this experiment, 5×104HeLa cells were seeded to eachwell.Prior to siRNA treatment, cells were washed three times withDMEM without serum. For silencing, On Target Plus SmartPool Mouse NCX 1 (Thermo Fisher Scientific, USA) silencerwas used. As a scrambled control, the On Target Plus ControlPool Non-targeting pool (Thermo Fisher Scientific, USA) wasused. Transfection was done with the X-tremeGENE siRNAtransfection reagent (Roche Diagnostics, Germany), accordingto the manufacturer protocol. Cells with added siRNA wereincubated 4 h at 37 °C in serum-free medium. After 4 h, serumwas added to the cells and they were incubated for additional48 h. After first 24 h, GYY4137 was added to one group ofHeLa cells with silenced NCX 1 and cells were incubated foradditional 24 h. All groups of cells were then harvested and usedin further experiments.

Western blot analysis

Cells were scraped and pelleted at 1,000×g for 5 min. Pelletwas resuspended in 10 mM Tris–HCl, pH 7.5, 1 mMphenylmethyl sulfonylfluoride (Serva, Heidelberg, Germany),protease inhibitor cocktail tablets (complete EDTA-free, RocheDiagnostics, Mannheim, Germany) and subjected to centrifu-gation for 10 min at 10,000×g at 4 °C. The pellet was resus-pended in Tris buffer containing 50 μM CHAPS (3-[(3-cholamidopropyl) dimethyl-ammonio] 1-propanesulfonate;Sigma, USA) and then incubated for 10 min at 4 °C. Proteinconcentration of lysate was determined by themethod of Lowry[34]. Twenty to 50 micrograms of protein extract from eachsample was separated by electrophoresis on 4–16 % SDS–polyacrylamide gradient gels (Amersham Biosciences, UK),and proteins were transferred to the Hybond-PVDF membrane

Pflugers Arch - Eur J Physiol

using semidry blotting (Amersham Biosciences, UK).Membranes were blocked in 5 % non-fat dry milk in Tris-buffered Saline with Tween-20 (TBS-T) for overnight at 4 °Cand then incubated for 1 h with appropriate primary antibody.Following washing, membranes were incubated with second-ary antibodies to mouse or rabbit IgG conjugated to horseradishperoxidase (Amersham Biosciences, UK), for 1 h at roomtemperature. An enhanced chemiluminiscence detection system(ECL Prime Western Blotting Detection Reagent, AmershamBiosciences, UK) was used to detect bound antibody.Glyceraldehyde-3-phosphate dehydrogenase (GAPDH,Abcam, Cambridge, UK, cat no. ab8245) signal was used asa housekeeper and control protein for quantification. Opticaldensity of individual bands was measured on Kodak Imagestation 440 device and quantified using PCBAS 2.0 software.

Antibodies raised against the following proteins were used:mouse monoclonal antibody to NCX1 (Abcam, Cambridge,UK, cat no. ab2869), a rabbit polyclonal antibody to β1-AR(Santa Cruz Biotechnology Inc., cat no. sc-568) and rabbitpolyclonal antibody to β2-AR (Abnova, USA, cat no.PAB2838). For β3-AR binding protein staining, a polyclonalantibody to β3-AR was used (Alpha Diagnostic International,USA, cat no.B3AR13-S). For AdCy7 binding protein staining,a polyclonal antibody to AdCy7 was used (Antibodies-online,DE, cat no. ABIN493201).

Measurement of the NCX activity

NCX1 activity was measured similarly as in Convery andHancox [7]. Plated cells were washed for 3 min with hypotoniccell solution (0.1 mMMgCl2 and 3 mM HEPES, pH 7.4), andthen, isotonic cell solution was added with Fluo-3 AM(Invitrogen, USA) and calcium (1 mM MgCl2, 10 mMHEPES pH 7.4, 10 mM glucose, CaCl2 to the final concentra-tion of 500 μM, Fluo-3 AM to the final concentration of 8 μMand pluronic acid to 0.04 %). After 10 min of loading in thedark at room temperature, cells were washed with isotonic cellsolution and solution of 5 mM KCl or 140 mM NaCl wasadded. The fluorescence signal was measured on fluorescencereader BioTek (BioTek Instruments, Inc.) at 37 °C. Excitationwas measured at 485 nm and emission at 528 nm. Calciumtransport was calculated from the difference in fluorescentsignal between KCl and NaCl solution. The control of fluores-cent signal was performed by adding of 10 % SDS and 50 mMEGTA pH 7.4 at the end of measurements. Calcium transportwas expressed as fluorescent arbitrary units.

Immunofluorescence

Immunofluorescence was performed as described in Pacakand coworkers [43]. Briefly, cells were fixed in ice-coldmethanol. Non-specific binding was blocked by incubationwith phosphate-buffered saline (PBS) containing 1 % bovine

serum albumin (Merck Biosciences, Darmstadt, Germany) for30 min. at 37 °C. Then, cells were incubated with primaryantibodies diluted 1:1,000 for 60 min at 37 °C. We have usedprimary mouse monoclonal antibody to NCX1 (Abcam,Cambridge, UK, cat no. ab2869), rabbit polyclonal antibodyto β1-AR (Santa Cruz Biotechnology, cat no. sc-568), andrabbit polyclonal antibody to β3-AR (Alpha DiagnosticInternational, cat no. B3AR13-S). Coverslips were washedin 0.02 % Tween/PBS and incubated with Alexa Fluor 594Goat Anti Mouse IgG (H + L) secondary antibody (LifeTechnologies, USA) and CFTM488 Goat Anti-Rabbit IgG(H + L) (Biotium, Hayward, CA, USA) secondary antibodyfor 60 min at 37 °C. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; 1:36,000, Invitrogen, Life technolo-gies, USA). Finally, cells weremounted onto slides inmountingmedium with Citifluor (Agar Scientific Ltd., Essex, UK), ana-lyzed by Zeiss LSM510 laser scanning confocal microscopysystem mounted on a Zeiss Axiovert 200 M inverted micro-scope. Images were taken with a Plan Apochromat 40×/1.3immersion oil objective. Images were scanned at scan speed 7(260 Hz line frequency), 1,024×1,024 pixels, 12-bit data depthwith an average mode 4× line at optical zoom 2. All images tobe compared were taken at the same settings.

Immunoprecipitation

Appropriate monoclonal antibodies (3 μg) or polyclonal anti-bodies (~6 μg) were incubated with ~2×107 (50 μl) washedmagnetic beads (Dynabeads M-280; coated with goat anti-mouse IgG or M-280 sheep anti-rabbit IgG; Invitrogen DynalAS, Norway) for overnight at 4 °C on a rotator (VWRInternational). As negative controls, beads were incubated withmouse IgG1κ (MOPC-21; Sigma, USA). The beads with at-tached antibody were washed (twice, 200 μl) with phosphate-buffered saline (PBS). Proteins were immunoprecipitated from1mg of detergent-extracted total protein by incubation for 4 h at4 °C with antibody-bound beads. Following incubation, thesupernatant was frozen and later used for immunoblotting.Bead complexes were washed with (four times 200 μl) PTA(1,45 M NaCl, 0,1 M NaH2PO4, 0,1 M sodium azide, and0.5 % Tween 20, pH 7.0). Immunoprecipitated proteins werethen extracted with 60 μl of 2× sodium dodecyl sulfate (SDS)sample buffer (0.5 M Tris, 10 % SDS, glycerol, bromphenolblue, DTT) and boiled for 5 min.

cAMP assay

The cAMP assay was performed using commercially availablekit according to the manufacturer’s recommendations (cAMPDirect Immunoassay Kit, BioVision Incorporated, CA, USA).Briefly, samples were added to the Protein G coated 96-wellplate cAMP-horseradish (HRP) conjugate directly competeswith cAMP from sample binding to the cAMP antibody on

Pflugers Arch - Eur J Physiol

the plate. After incubation and washing, the amount of cAMP-HRP bound to the plate was determined by reading HRPactivity at OD450 nm. The intensity of OD450 nm is inverselyproportional to the cAMP concentration in samples.

Microarray assay

This experiment was performed on PC12 cells. After theisolation, RNA was further purified using GeneJET™ RNAPurification Kit (ThermoScientific, USA) to eliminate co-purified contaminants. The purity and concentration weremeasured on NanoDrop 2000 (ThermoScientific, USA).Integrity of RNA was check using BioAnalyzer RNA 6000Nano Kit (Agilent Technologies, USA). RNA samples withpurity ratio (of both 260/A280 and 260/230) higher than 2 andwith RNA integrity numbers (RIN) higher than 8 were pro-ceed to sample labeling reaction. Sample labeling reactionswere performed using Quick Amp Labeling Kit (AgilentTechnologies, USA). Briefly, 200 ng of total RNA wereprimed with T7-promoter primer and cDNAwere synthesizedusing moloney murine leukemia virus reverse transcriptase. Innext step, T7 polymerase incorporates Cy3-or Cy5-labeledCTP in amplification process and thus generates labeledcRNA. During amplification and labeling control, RNAs fromRNA Spike-In kit (Agilent Technologies, USA) were addedinto reaction. Target after labelingwas purified to remove non-incorporated nucleotides and reaction components usingGeneJET™ RNA Purification Kit (ThermoScientific, USA).Amplified and labeled target’s concentration, purity, and spe-cific activity were determined using NanoDrop 2000 modelMicroarray (ThermoScientific, USA). Labeled samples(825 ng), Cy5-labeled treated sample and Cy3-labeled appro-priate control sample, were combined together and, in hybrid-ization mix reaction (Gene Expression Hybridization Kit,Agilent Technologies, USA), were incubated exactly 30 minat 60 °C to fragment cRNA. Fragmented samples were addedonAgilent HumanGene Expression 4×44 k v3Microarray andhybridize 17 h at 65 °C by rotating slide at rotate speed 10 rpm.Slides were washed in Wash buffers 1 and 2 (Gene ExpressionWash Buffer Kit, Agilent Technologies, USA) according to themanufacturer’s instructions and scanned using Agilent scannerat 5-μm resolution.

Image processing was performed automatically usingFeature Extraction Software 11.5 (Agilent Technologies,USA), and acquired files containing signal intensities of Cy5and Cy3 from spots, which represent specific probes, wereimported into GeneSpring GX software to analyze differencein gene expression of particular genes between treated andcontrol sample. During the evaluation, only detected flags(significantly different from background) were proceeded toanalysis. Level of gene expression was determined, and sig-nificant differences were considered if fold of change betweentreated and control samples was ≥2.0. Pathway analysis

module was applied to revealed molecular pathways signifi-cantly affected in our experiment (p ≤0.05).

Detection of apoptosis with Annexin-V-FLUOS

HeLa cells were trypsinized, washed with PBS, and pelleted1,000×g for 5 min. Cell pellet from each well was resuspend-ed in 200 μl of Annexin-V FLUOS (Roche Diagnostics,Switzerland) labeling solution and incubated at room temper-ature in dark for 20 min. Labeling solution included incuba-tion buffer with 10 mMHEPES/NaOH pH 7.4, 140 mMNaCland 5 mM CaCl2, 2 μl of Annexin-V-FLUOS and 0.02 μgpropidium iodide (Roche Diagnostics, Switzerland). After theincubation, reaction was stopped by adding 300 μl ice-coldPBS and measured in 96-well plates on Accuri C6 flowcytometer (BD Accuri Cytometers Ann Arbor, MI, USA).

Statistical analysis

The results are presented as mean ± SEM. Each value repre-sents an average of at least 6 wells from at least three inde-pendent cultivations of HeLa cells. Statistical differencesamong groups were determined by ANOVA. Statistical sig-nificance of p <0.05was considered as significant for *(+) and**(++) p <0.01, ***(+++) p <0.001. For multiple compari-sons, an adjusted t test with p values corrected by theBonferroni method was used (Instat, GraphPad Software).

Results

After 24 h treatment of HeLa cells with the GYY4137, weobserved rapid increase in the NCX1 mRNA (Fig. 1a), proteinlevels (Fig. 1b) and also in the NCX activity (Fig. 1c). Usingimmunofluorescence with the NCX1 antibody, a strong specificsignal appeared in the group of cells treated with GYY4137 (G)compared to control cells (cont). No signal was observed innegative control (NC; Fig. 1d), where primary antibody wasomitted. When HeLa cells were treated simultaneously with theGYY4137 and DTT (GDTT), GYY4137 induced increase inthe NCX1 mRNA (Fig. 2a), protein (Fig. 2b), and activity(Fig. 2c) diminished. When HeLa cells were treated only withDTT, any significant difference compared to control cells (cont)was observed in themRNA, protein, and activity levels (Fig. 2).Thus, DTT was able to prevent completely GYY-induced in-crease in the NCX1 mRNA (Fig. 2a), protein (Fig. 2b), andactivity (Fig. 2c).

Gene profiling with the microarray chip revealed increasein the gene expression of adenylyl cyclase 7 (Fig. 3a; numbersshows fold of increase (red) or decrease (green) compared tocontrols), but decrease in the gene expression of adenylylcyclase 8. Increase in AdCy7 mRNA was verified by real-time PCR (Fig. 3b, upper graph) and increase in AdCy7

Pflugers Arch - Eur J Physiol

protein was verified by Western blot analysis (Fig. 3b, lowergraph). Since from these results nothing can be concludedabout the cAMP levels, we measured cAMP concentration incontrol, GYY4137 treated cells, but also in GYY4137 treatedcells, where NCX1 was silenced (GsiN), or scrambled siRNA(Gscr) was added (Fig. 3c). We observed almost two timesincrease in cAMP concentration in GYY4137 treated cellscompared to control, untreated cells. When NCX1 was si-lenced, GYY4137-induced increase did not appear (Fig. 3c).Since scrambled siRNA did not cause any effect, we considerdecreased cAMP due to NCX1 silencing to be a specific. Thisexperiment suggests at least communication with the β-ARsdue to the sulfide signaling. Thus, we measured changes in

gene expression (Fig. 4, upper lane) and protein levels (Fig. 4,lower lane) of the β1, β2, and β3 ARs due to the GYY4137(G) treatment for 24 h. We observed increase in mRNA andalso in the protein of β1 and β3, but not the β2 ARs afterGYY4137 treatment (Fig. 4). In further experiments, we triedto prove the mutual interaction of the β1 and β3 ARs andNCX1. Thus, we performed immunoprecipitation experi-ments (Fig. 5) and also immunofluorescence with doublestaining (Fig. 6). Firstly, we immunoprecipitated the β1 ARs(Fig. 5a) and/or β3 receptors (Fig. 5b) from control andGYY4137 treated cells and hybridized them with antibodiesagainst NCX1, β1, and β3 ARs. We compared either crudehomogenates (H) or immunoprecipitates (IP). In order to

**

Cont G NC

cont G0

5

10

15

20

25

30

NC

X1p

rote

in/G

AP

DH

(a.

u.)

***

cont G0

100

200

300

400

500

600

700

800

NC

X1a

ctiv

ity (

a.u.

)

***a b c

d

cont G0

40

80

120

160

200

NC

X1m

RN

A/c

yclo

mR

NA

(a.

u.)

cont G

NCX1GAPDH

Fig. 1 Effect of the slow sulfide donor GYY4137 (G; 10 μM) on theNCX1 mRNA (a), protein (b) and activity (c) levels, as well as immuno-fluorescence with the NCX1 antibody (d) of GYY4137 (G) treated cellscompared to control (cont), untreated HeLa cells. GYY4137 increasedmRNA (a), protein (b) and activity (c) of the NCX1. Above the graph aa typical melting curve is shown. For protein (b), typical gel of the NCX1and GAPDH protein is shown in the upper part of the graph. In d, NC

serves as a negative immunofluorescence control, where primary antibodywas omitted. NC proves a specificity of the signal. Columns are displayedas mean ± SEM, and each value represents an average of at least threeindependent cultivations, each performed in triplicates. Statistical signifi-cance compared to controls **p<0.01 and ***p<0.001. Immunofluores-cence showed also the higher signal of the NCX1 in GYY4137 treatedcells, but any translocation compared to control, untreated cells

Pflugers Arch - Eur J Physiol

verify specificity of immunoprecipitated ARs, we performednegative controls (NC), where protein was not added. NCX1signal was quantified and is shown in column graph (Fig. 5c, c),where striped columns represent NCX1 signal from homoge-nates and black columns represent NCX1 signal from immu-noprecipitates. In both groups, we observed rapid increase inthe β1 and/or β3 signal in group of GYY-treated cells com-pared to control group of cells, which suggests the mutualinteraction of the β1 and β3 with the NCX1. Also, immuno-fluorescence with both β1 (green) and NCX1 (red; Fig. 6,upper part) and β3 (red) and NCX1 (green; Fig. 6, lower part)showed co-localization (yellow) of both these ARs with theNCX1.

Because of known effect of an adrenergic modulation onthe NCX, NCX1 mRNA, protein, and NCX activity wereevaluated in the presence of a nonspecific β1 and β2 blockerpropranolol hydrochlorid (P), specificβ2 blocker ICI 118,551hydrochlorid (ICI), and specific β3 blocker SR 59230 A (SR;Fig. 7). We have shown that in the presence of GYY4137,propranolol (GP) and SR (GSR) decreased mRNA (Fig. 7a),protein (Fig. 7b), and activity (Fig. 7c) of the NCX1. Specificβ2 blocker ICI had no effect on the NCX1mRNA and protein(Fig. 7a, b); therefore, we did not measure activity of the NCXafter ICI treatment. By the same way, we also determined the

effect of specific α1 blocker doxazosine on GYY4137 treatedcells. Since we did not observed any changes in mRNA,protein, and activity level (not shown), we propose that α1AR are not involved in GYY4137 induced NCX1 increase.Finally, GYY4137 treatment for 24 h increased apoptosis in aconcentration dependent manner (Fig. 8a). GYY4137 inducedapoptosis was partially prevented by nonspecific β1/β2blocker propranolol (P) and completely prevented by thespecific β3 blocker SR 59230 A (SR) (Fig. 8b). Specific β2blocker ICI did not affect apoptosis in GYY4137 treated HeLacells for 24 h (Fig. 8b). In order to ensure that partial preven-tion in the GYY4137 induced apoptosis by propranolol is dueto the blockade of β1 AR, we performed the same experimentusing specific β1-blocker CGP-20712A (CGP) (Fig. 8c). Weobserved the same partial decrease in the GYY4137 inducedapoptosis, which point to the partial involvement ofβ1 ARs inthis process (Fig. 8c). In order to verify also a participation ofthe NCX1 in the GYY4137 induced apoptosis, we measuredapoptosis induction in the group of cells, where NCX1 wassilenced (to 70 %; Fig. 8d, e). As a control of the specificity,scrambled siRNAwas used. GYY4137 induced apoptosis wascompletely prevented in cells, where NCX1 was silenced, butnot in the group of HeLa cells treated with scrambled siRNA(Fig. 8d, e).

a b c

*** ******+++++++

NCX1

GAPDH

NCX1

β-actin

cont G DTT GDTT0

2

4

6

8

10

NC

X1

mR

NA

/β a

ctin

mR

NA

(a.

u.)

cont G DTT GDTT0

200

400

600

800

NC

X1

activ

ity (

a.u.

)

cont G DTT GDTT0

5

10

15

20

25

30

NC

X1

prot

ein/

GA

PD

H p

rote

in (

a.u.

)

Fig. 2 Effect of 24-h treatment with the DTT (10 μM) on GYY4137 (G ;10 μM) induced increase in the NCX1 mRNA (a), protein (b), and NCXactivity (c). In the presence of DTT, no significant increase in NCX1mRNA, protein, and activity occurs in GYY4137 treated cells (GDTT).In the upper part of graphs a and b , typical gel of the NCX1 and ahousekeeper is shown. For the PCR, β-actin was used as a housekeeper.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is shown as ahousekeeper in Western blots. Each column represents mean ± SEMand is an average of at least three independent cultivations. Gels representtypical result for each protein. Statistical significance compared to con-trols ***p <0.001 and GYY4137 (G )-treated group vs. GYY/DTT(GDTT)-treated group ++p<0.01 and +++p <0.001

Pflugers Arch - Eur J Physiol

Discussion

Great potentials of H2S for dealing with different pathologicalsituations inspire great efforts for exploring the best ways todeliver H2S and to invent H2S-based strategies [61]. Sucheffort is dedicated in studies of some neurological and cardio-vascular diseases, inflammation, but also in cancer. Resultsobtained up to now differ, since therapeutic effectivenessdepends on several factors, e.g., rate of H2S delivery, amountof H2S delivered to the cells, and type of cells. Therefore,studies using different H2S donors in variety of cells mighthelp to unravel the signaling potential of H2S and target itpotentially to a therapeutic exploitation.

In this work, we revealed that a slow H2S donor GYY4137increased expression of the NCX1, β1, and β3 ARs, which

form complex and are able to induce apoptosis in HeLa cells.Besides NCX1, some other calcium transport systems are alsomodulated by H2S–IP3 receptors [30], L-type calcium chan-nels [49], etc. Changes in these transport systems on themRNA and protein levels or activity can affect intracellularcalcium signaling [35, 44, 53]. It is widely accepted thatsulfide signaling increases the intracellular calcium. In ourrecent article [30], we have also shown that in HeLa cells,24-h GYY4137 treatment mobilizes cytosolic calcium in aconcentration dependent manner. In endothelial cells, H2Swas shown to stimulate Ca2+ influx by recruiting reversemode of the NCX and KATP channels [37]; nevertheless,our results provide the first evidence showing that H2S affectsgene expression, protein level, and activity of the NCX1 incancer cells.

a b c

cont G0

1

2

3

4

5

6A

dCy7

pro

tein

(a.

u.)

** **

cont G GsiN Gscr0

1

2

3

4

5

6

cAM

P(p

mol

/ng

prot

ein)

cont G0

1

2

3

4

AdC

y7 m

RN

A/c

yclo

mR

NA

***

**

cont G

AdCy7

GAPDH

Fig. 3 Adenylate cyclase 7 (AdCy7) and cAMP determination in controland GYY4137 (G)-treated cells. By the method of gene profiling onmicrochips (Agilent), we have clearly shown that Adcy 7 is upregulatedand Adcy8 is downregulated in HeLa cells due to GYY4137 treatment(a). Increase in the AdCy7 mRNA (b , upper graph) and AdCy7 protein(b , lower graph ) proved the effect of G. Level of cAMP (c ) wasincreased approximately two times due to G treatment compared to

controls (cont) and remained on the control levels, when GYY4137was applied to the cells with silenced NCX1 (GsiN) for 48 h. HeLa cellswith scrambled siRNA remained increased due to the GYY4137 treat-ment (Gscr). Each column is displayed as mean ± SEM and represents anaverage of at least four independent cultivations. Statistical significancecompared to controls **p <0.01

Pflugers Arch - Eur J Physiol

H2S-induced NCX1 increase was observed on the mRNAlevel; thus, pointing to the H2S modulation on the transcrip-tional level. Multiple tissue specific variants of the Ncx1 geneexist due to the alternative promoter usage (H1, K1 and Br1)and also alternative splicing [4]. Ncx1 gene is regulated by avariety of transcription factors, including AP1 protein, AP2protein, cAMP-response element-binding protein (CREB),NF-κB, and HIF1. NF-κB dependent NCX1 upregulationmay play a fundamental role in calcium refilling into theendoplasmic reticulum in cortical neurons [52]. β-Adrenergic receptor-simulated Ncx1 upregulation might bemediated via activation of the AP1-factor [36]. Also, HIF-1α is involved in hypoxia-induced upregulation of the NCX1[24]. Based on the current knowledge, we proposed that β-adrenergic modulation and thus increased cAMP might beinvolved through the CREB. In our study, the gene profilingrevealed that adenylyl cyclase 7 is upregulated and adenylylcyclase 8 is downregulated due to GYY4137 treatment for

24 h. However, cAMP levels were increased in HeLa cells,which is in agreement with observations on endothelial cells[39] and neuronal and glial cells [53]. Also, some other papersreported increase in the cAMP levels due to sulfide signaling[1, 51]. On the other hand, H2S was also shown to decreasecAMP levels [63]. Increase in the cAMP levels suggests aninvolvement of β-ARs. We showed that β1 and β3-ARs, butnot the β2 ARs, are upregulated by GYY. Yong and co-workers [65] showed that in the rat hearts H2S may negativelymodulate beta-adrenoceptor function via inhibiting adenylylcyclase activity. Nevertheless, we observed that both thesereceptors co-precipitate with the NCX1, thus forming a β1-AR/β3-AR/NCX1 complex. This complex was involved in anapoptosis induction. We hypothesize that β1-AR/β3-AR/NCX1 complex might not only increase but also change thecalcium flux through the NCX1 from outward to the inward(reverse) mode. Increased calcium concentration activates IP3receptors, which releases calcium from the endoplasmic

cont G0

4

8

12

16

20

24

β1-A

R p

rote

in (

a.u.

)

cont G0

2

4

6

8

β2-A

R p

rote

in (

a.u.

)

cont G0

4

8

12

16

20

24

β3-A

Rpr

otei

n (a

.u.)

**

**

***

*

cont G0

4

8

12

16

20

β1-A

R m

RN

A/c

yclo

mR

NA

(a.

u.)

cont G0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

β2-A

R m

RN

A/c

yclo

mR

NA

(a.

u.)

cont G0

5

10

15

20

25

30

35

β3-A

R m

RN

A/c

yclo

mR

NA

(a.

u.)

β1 AR

β2 AR

β3 AR

β1 AR

β2 AR

β3 AR

GAPDH

GAPDH

GAPDH

Fig. 4 Effect of the slow sulfide donor GYY4137 (G ; 10 μM) on theβ1,β2, and β3-adrenergic receptor (AR) mRNA (upper lane) and protein(lower lane) compared to control (cont), untreated HeLa cells. GYY4137increased mRNA and protein of the β1 and β3, but not β2-ARs. On theright side, typical melting curves are shown. For protein, typical gels are

shown in the right side of the bottom line of the figure. GAPDH has beenused as a housekeeper in Western blot studies. Columns are displayed asmean ± SEM and each value represents an average of at least threeindependent cultivations, each performed in triplicates. Statistical signif-icance compared to controls *p<0.05, **p <0.01 and ***p <0.001

Pflugers Arch - Eur J Physiol

cont GH IP NC H IP NC

β1-AR

β1-AR

cont GH IP H IP

NCX1

**

*

cont GH IP NC H IP NC

β3-AR

β3-AR

cont GH IP H IP

NCX1

*

*

β1-ARs immunoprecipitation

β3-ARs immunoprecipitation

a

b

c

d

cont GYY0

100

200

300

400

500

600

NC

X1

pro

tein

(a.

u.)

cont GYY0

2000

4000

6000

8000

10000

NC

X1

prot

ein

(a.u

.)

Fig. 5 Immunoprecipitation with either β1-AR antibody (a) or β3-ARantibody (b). Immunoprecipitates (IP) were subsequently loaded on a geland hybridize with NCX1 and β3-AR, or NCX1 and β1-AR. As acontrol, crude homogenates (H ; 20 μg per lane) were loaded on a samegel.NC negative controls. Amounts of NCX1 signals were quantified andare shown as graphs (c , d). Striped columns represent NCX1 signal from

homogenates, black columns represent NCX1 signal from immunopre-cipitate with either β1-AR antibody (c) orβ3-AR antibody (d). Columnsare displayed as mean ± SEM, and each value represents an average of atleast three independent cultivations. Statistical significance compared tocontrols *p <0.05 and **p <0.01

Cont G NC

β1-AR

β3-AR

Fig. 6 Determination of theNCX1, β1-ARs and β3-ARs inHeLa cells by theimmunofluorescence. Nucleiwere labeled with DAPI and arevisualized as blue signal.Immunofluorescence with bothβ1 (green ; upper part) and NCX1(red ; upper part) and β3 (red;lower part) and NCX1 (green ;lower part) showed co-localization (yellow) of both theseARs with the NCX1. Negativecontrols (NC) proved a specificityof the signal

Pflugers Arch - Eur J Physiol

reticulum. This calcium is known to enter mitochondria [20],which cause release of cytochrome C and activation of theinner apoptotic pathway. Involvement of the β-ARs in theapoptosis induction depends probably on the type of cancercells. Zhang and co-workers [66] presented experimental ev-idence that strongly supports the antitumor and anti-invasioneffects of inhibiting β1-or β2-adrenergic receptors in MIAPaCa-2 and BxPC-3 pancreatic cancer cells. He suggested thatinhibition of β1- or β2-adrenergic receptors could be aneffective approach for the inactivation of CREB, AP-1,NFκB and its target genes, such as MMP-9, MMP-2, andVEGF expression, and for further inhibition of cancer cellinvasion and proliferation.

GYY4137 was reported to release H2S slowly and steadily,either in aqueous solution or administered to the animal [32].The rate of H2S release from GYY4137 (1 mmol/L, i.e.,100 nmol incubated) was 4.17±0.5 nmol/25 min, i.e., ~4 %/25 min. When incubated in aqueous buffer (pH 7.4, 37 °C),H2S release sustained over 75 min and after administration(intravenous or intraperitoneal) of GYY4137 to anesthetized

rats, plasma H2S (defined as H2S, HS–, and S2–) concentration

was increased at 30 min and remained elevated over the180 min [32]. From these reported results, we might estimatethat in our study the free concentration of H2S released fromGYY4137 was <1 μmol/l and this might be similar to thephysiological concentrations. Thus, although we measured theexogenous effect of the H2S, we might speculate that if underphysiological conditions endogenous H2S reaches similar con-centration, the effect on ARs and NCX1 might be the same.

H2S appears to signal predominantly by S-sulfhydratingcysteines in its target proteins, what changes their function[45]. Hydrogen sulfide anion (HS−) regulates the metabolismand signaling actions of various electrophiles. It reacts withelectrophiles via direct sulfhydration and modulates cellularredox signaling [42]. Several proteins, including NF-κB, werereported to be regulated by S-sulfhydration; however, a pos-sible sulfhydration of proteins influenced by H2S in our studyis still unknown.

Apoptosis is the dominant feature in a cancer treatment. Inour experiments, H2S-induced activation of the NCX1

a b ccont G GP GSR GICI

cont G GP GSR GICI0

2

4

6

8

NC

X1

mR

NA

/cyc

lo m

RN

A (

a.u.

)

cont G GP GSR0

200

400

600

800

NC

X1

activ

ity (

a.u.

)

cont G GP GSR GICI0

10

20

30

40

50

60

NC

X1p

rote

in (

a.u.

)**

**+++

++++

+++

*

NCX1

GAPDH

Fig. 7 Effect of the beta-blockers in the presence of the slow sulfidedonor GYY4137 (G ; 10 μM) on the NCX1 mRNA (a), protein (b), andactivity (c) levels. GYY4137 increased mRNA (a), protein (b), andactivity (c) of the NCX1. This increase was significantly blocked by thenonspecific β1–β2 blocker propranolol hydrochloride (GP), and also bythe specific β3 blocker SR 59230A (GSR). Specific β2 blocker ICI 118,551 hydrochloride (GICI) did not prevent GYY4137 induced increase of

the NCX1 mRNA and protein. For the NCX1 protein determination (b),typical gel for both, NCX1 and GAPDH protein is shown in the upperpart of the graph. Columns are displayed as mean ± SEM and each valuerepresents an average of at least three independent cultivations, eachperformed in triplicates. Statistical significance compared to controlsrepresents *p <0.05 and **p <0.01. Statistical significance compared toGYY treated group represents +p <0.05 and ++p <0.01

Pflugers Arch - Eur J Physiol

resulted in the increase of apoptosis. Ability of the NCX1 toinduce apoptosis was already demonstrated in motor neuronsin adult spinal cord slices [38]. Also, NCX overexpressioninduces endoplasmic reticulum-related apoptosis and caspase-12 activation in insulin-releasing BRIN-BD11 cells [9].Nevertheless, both anti-apoptotic and pro-apoptotic effectswere ascribed to the H2S. Anti-apoptotic effect of the H2Swas described mainly on neuronal cells [57, 58]. Zhang andcoworkers [66] showed that NaHS inhibited the apoptosis ofrat hippocampus neurons induced by vascular dementia. Apro-apoptotic effect of H2S was firstly reported on vascularsmooth muscle cells [62]. From that time, pro-apoptotic effectof H2S was confirmed in epithelial cells [25], isolated mousepancreatic acini [6] and also in some cancer cell lines, e.g.,PC12 cell line [30]. Differences in the response of cells to H2Sin respect to apoptosis might lie in the cell type specificity. It

was already shown that H2S and H2S prodrugs seem to becapable of inhibiting all stages in cancer development [48].Thus, H2S appears to exert powerful pro-apoptotic actions oncancer cells of different origins, which suggests that H2S pro-drugs might be developed into effective anticancer agents.Indeed, in HeLa cells we observed significant increase inapoptosis due to the GYY4137 treatment, which was not seenin cells, where NCX1 was silenced. This would strongly pointto an involvement of the NCX1 in the process of apoptosis.

In summary, we have shown that in HeLa cells, sulfidesignaling for 24 h increased not only NCX1 but also β1 andβ3 ARs. These proteins co-localize and forms complex,which is able to increase apoptosis in these cells. Inhibitionsof each of these proteins result in prevention of GYY4137-induced apoptosis in HeLa cells. These results support theimportance of sulfide signaling in the apoptosis induction and

Cont GsiN

G Gscr

cont G1 G10 G1000

5

10

15

20

25

30

Ann

exin

V p

ositi

ve c

ells

(%

)

cont G GsiN Gscr0

4

8

12

16

20

Ann

exin

V p

ositi

ve c

ells

(%

)

a b c

d e

**

***

cont G CGP GCGP0

5

10

15

20

Ann

exin

V p

ositi

ve c

ells

(%

)

*

******

**

cont G GP GSR GICI0

5

10

15

20

25

Ann

exin

V p

ositi

ve c

ells

(%

)

***

+

+++++

*

+

***

Fig. 8 Ability of the slow sulfide donor GYY4137 to induce apoptosis inHeLa cells was measured by Annexin V assay. Number of Annexin Vpositive cells increased by the concentration-dependent manner (a), whencells were treated by 1, 10, and 100 μMGYY4137. Number of Annexin V-positive cells were partially decreased in HeLa cells treated with GYY4137and propranolol (GP; b) and decreased to a higher extent in cells treatedwith GYY4137 and SR 59230A (GSR; b) compared to GYY4137 treatedcells only (G; b). No decrease compared to G was observed in the cellstreatedwith ICI 118,551 hydrochloride (GICI; b). Sincewewanted to proofthat beta 1 blocking causes only slight decrease in the Annexin V positive

cells compared to G, we used specific β1 blocker CGP 20712A (GCGP;c).We observed the same effect as with propranolol. Also, whenHeLa cellswith silenced NCX1 (GsiN) were subjected to G, GYY4137 inducedincrease in Annexin V-positive cells was completely prevented (d). Forthe illustration, Annexin V plots for the experiment (d) are shown in part e.Columns are displayed as mean ± SEM and each value represents anaverage of at least three independent cultivations, each performed in tripli-cates. Statistical significance compared to controls represents *p<0.05,**p <0.01, and ***p <0.001 and statistical significance compared toGYY4137 represents +p<0.05, ++p<0.01, and +++p<0.001

Pflugers Arch - Eur J Physiol

might have an impact on the H2S involvement in the potentialtreatment strategy of cancer.

Acknowledgments This work was supported by grants APVV 51-0045-11, VEGA 2/0095/13 and Center of Excellence for Studying Met-abolic Aspects of Development, Diagnostics and Treatment of the Onco-logic Diseases (CEMAN). Project implementation, DNA-DG: ITMS26240220058, was supported by the Research & Development Opera-tional Programme funded by the ERDF.

References

1. Abe K, Kimura H (1996) The possible role of hydrogen sulphide asan endogenous neuromodulator. J Neurosci 16:1066–1071

2. Andrikopoulos P, Baba A, Matsuda T, Djamgoz MB, Yaqoob MM,Eccles SA (2011) Ca2+ influx through reverse mode Na+/Ca2+ ex-change is critical for vascular endothelial growth factor-mediatedextracellular signal-regulated kinase (ERK) 1/2 activation and angio-genic functions of human endothelial cells. J Biol Chem 286:37919–37931

3. Bai Y, Morgan EE, Giovannucci DR, Pierre SV, Philipson KD,Askari A, Liu L (2013) Different roles of the cardiac Na+/Ca2+−exchanger in ouabain-induced inotropy, cell signaling, and hyper-trophy. Am J Physiol Heart Circ Physiol 304:H427–H435

4. Barnes KV, Cheng G, Dawson MM, Menick DR (1997) Cloning ofcardiac, kidney, and brain promoters of the feline Ncx1 gene. J BiolChem 272:11510–11517

5. Baskar R, Sparatore A, Del Soldato P, Moore PK (2008) Effect of S-diclofenac, a novel hydrogen sulfide releasing derivative in-hibits rat vascular smooth muscle cell proliferation. Eur JPharmacol 594:1–8

6. Cao Y, Adhikari S, Anq AD, Moore PK, Bhatia M (2006)Mechanism of induction of pancreatic acinar cell apoptosis by hy-drogen sulfide. Am J Physiol Cell Physiol 291:C503–C510

7. Convery MK, Hancox JC (2000) Na+–Ca2+ exchange current fromrabbit isolated atrioventricular nodal and ventricular myocytes com-pared using action potential and ramp waveforms. Acta PhysiolScand 168:393–401

8. Cuozzo F, Raciti M, Bertelli L, Parente R, Di Renzo L (2012) Pro-death and pro-survival properties of ouabain in U937 lymphomaderived cells. J Exp Clin Canc Res 15:31–95

9. Diaz-Horta O, Kamagate A, Herchuelz A, Van Eylen F (2002) Na/Caexchanger overexpression induces endoplasmic reticulum-related ap-optosis and caspase-12 activation in insulin releasing BRIN-BD11cells. Diabetes 51:1815–1824

10. Diaz-Horta O, Van Eylen F, Herchuelz A (2003) Na/Ca exchangeroverexpression induces endoplasmic reticulum stress, caspase-12release, and apoptosis. Ann New York Acad Sci 1010:430–432

11. Elefteriou F, Campbell P, Ma Y (2013) Control of bone remodelingby the peripheral sympathetic nervous system. Calcif Tissue Int. doi:10.1007/s00223-013-9752-4

12. Fan HN, Wang HJ, Yang-Dan CR, Ren L, Wang C, Li YF, Deng Y(2012) Protective effects of hydrogen sulfide on oxidative stress andfibrosis in hepatic stellate cells. Mol Med Rep. doi:10.3892/mmr.2012.1153

13. Fernandes VS, Ribeiro AS, Barahoma MV, Orensanz LM, Martinez-Saenz A, Recio P, Martinez AC, Bustamante S, Carballido J, Garcia-Sacristan A, Prieto D, Hernandez M (2013) Hydrogen sulfide medi-ated inhibitory neurotransmission to the pig bladder neck: role ofKATP channels sensory nerves and calcium signaling. J Urol. doi:10.1016/j.juro.2013.02.103

14. Frishman WH (2013) ß-Adrenergic blockade in cardiovascular dis-ease. J Cardiovasc Pharmacol Ther 18:310–319

15. Furne J, Saeed A, Levitt MD (2008) Whole tissue hydrogen sulfideconcentrations are orders of magnitude lower than presently acceptedvalues. Am J Physiol Regul Integr Comp Physiol 295:R1479–R1485

16. Golden KL, Fan QI, Chen B, Ren J, O’Connor J, Marsh JD (2000)Adrenergic stimulation regulates Na+/Ca2+ exchanger expression inrat cardiac myocytes. J Mol Cell Cardiol 32:611–620

17. Golden KL, Ren J, O’Connor J, Dean A, DiCarlo SE, Marsh JD(2001) In vivo regulation of Na/Ca exchanger expression by adren-ergic effectors. Am J Physiol Heart Circ Physiol 280:H1376–H1382

18. Goldhaber JI, Philipson KD (2013) Cardiac sodium–calcium ex-change and efficient excitation–contraction coupling: implicationsfor heart disease. Adv Exp Med Biol 961:355–364

19. Gu C, Ma YC, Benjamin J, Littman D, Chao MV, Huang XY (2000)Apoptotic signaling through the beta-adrenergic receptor. A new Gseffector pathway. J Biol Chem 275:20716–20733

20. Hanson CJ, Bootman MD, Roderick HL (2004) Cell signaling: IP3receptors channel calcium into cell death. Curr Biol 14:R933–R935

21. Hu LF, Lu M, Wu ZY, Wong PT, Bian JS (2009) Hydrogen sulfideinhibits rotenone-induced apoptosis via preservation of mitochondri-al function. Mol Pharmacol 75:27–34

22. Hudecova S, Kubovcakova L, Kvetnansky R, Kopacek J, PastorekovaS, NovakovaM,Knezl V, Tarabova B, Lacinova L, Sulova Z, Breier A,Jurkovicova D, Krizanova O (2007) Modulation of expression of theNa+/Ca2+ exchanger in a heart of rat and mouse under stress. ActaPhysiol 190:127–136

23. Hudecova S, Lencesova L, Csaderova L, Sedlak J, Bohacova V,Laukova M, Krizanova O (2013) Isoproterenol accelerates apoptosisthrough the over-expression of the sodium/calcium exchanger inHeLa cells. Gen Physiol Biophys 32:310–323

24. Hudecova S, Lencesova L, Csaderova L, Sirova M, Cholujova D,Cagala M, Kopacek J, Dobrota D, Pastorekova S, Krizanova O(2011) Chemically mimicked hypoxia modulates gene expressionand protein levels of the sodium calcium exchanger in HEK 293 cellline via HIF 1α. Gen Physiol Biophys 30:196–206

25. Imai T, Ii H, Yaegaki K, Murata T, Sato T, Kamoda T (2009) Oralmalodorous compound inhibits osteoblast proliferation. J Periodontal80:2028–2034

26. Ishigami M, Hiraki K, Umemura K, Ogasawara Y, Ishii K, Kimura H(2009)A source of hydrogen sulfide and a mechanism of its release inthe brain. Antioxid Redox Signal 11:205–214

27. Iwai-Kanai E, Hasegawa K, Araki M, Kakita T, Morimoto T,Sasayama S (1999) Alpha- and beta-adrenergic pathways differen-tially regulate cell type-specific apoptosis in rat cardiac myocytes.Circulation 100:305–311

28. Kurata Y, Hisatome I, Shibamoto T (2012) Roles of sarcoplasmicreticulum Ca2+ cycling and Na+/Ca2+ exchanger in sinoatrial nodepeacemaking: insights from bifurcation analysis of mathematicalmodels. Am J Physiol Heart Circ Physiol 302:H2285–H2300

29. Lee SW, Hu YS, Hu LF, Lu Q, Dawe GS,Moore PK,Wong PT, BianJS (2006) Hydrogen sulphide regulates calcium homeostasis inmicroglial cells. Glia 154:116–124

30. Lencesova L, Hudecova S, Csaderova L, Markova J, Soltysova A,Pastorek M, Sedlak J, Wood ME, Whiteman M, Ondrias K,Krizanova O (2013) Sulphide signalling potentiates apoptosisthrough the up-regulation of IP3 receptors type 1 and 2. ActaPhysiol 208:350–361

31. Lencesova L, Sirova M, Csaderova L, Laukova M, Sulova Z,Kvetnansky R, Krizanova O (2010) Changes and role ofadrenoceptors in PC12 cells after phenylephrine administration andapoptosis induction. Neurochem Int 57:884–892

32. Li L, Whiteman M, Guan YY, Neo KL, Cheng Y, Lee SW, Zhao Y,Baskar R, Tan CH, Moore PK (2008) Characterization of a novel,water-soluble sulfide-releasing molecule (GYY4137): new insightsinto the biology of hydrogen sulfide. Circulation 117:2351–2360

Pflugers Arch - Eur J Physiol

33. Liu YH, Lu M, Hu LF, Wong PTH, Webb GD, Bian JS (2012)Hydrogen sulfide in the mammalian cardiovascular system.Antioxid Redox Signal 17:141–185. doi:10.1089/ars.2011.4005

34. Lowry OH, Rosebrough HJ, Farr AL, Randall RJ (1951) Proteinmeasurement with the Folin phenol reagent. J Biol Chem 193:265–275

35. Maeda Y, Aoki Y, Sekiguchi F, Matsunami M, Takahashi T,Nishikawa H, Kawabata A (2009) Hyperalgesia induced by spinaland peripheral hydrogen sulfide: evidence for involvement of Cav3.2T-type calcium channels. Pain 142:127–132

36. Mani SK, Egan EA, Addy BK, Grimm M, Kasiganesan H,Thiyagarajan T, Renaud L, Brown JH, Kern CB, Menick DR (2010)Beta-Adrenergic receptor stimulated Ncx1 upregulation is mediatedvia a CaMKII/AP-1 signaling pathway in adult cardiomyocytes. J MolCell Cardiol 48:342–351

37. Moccia F, Bertoni G, Pla AF, Dragoni S, Pupo E, Merlino A,Mancardi D, Munaron L, Tanzi F (2011) Hydrogen sulfide regulatesintracellular Ca2+ concentration in endothelial cells from excised rataorta. Curr Pharm Biotechnol 12:1416–1426

38. Momeni HR, JarahzadehM (2012) Effect of a voltage sensitive calciumchannel blocker and a sodium–calcium exchanger inhibitor on apoptosisof motor neurons in adult spinal cord slices. Cell J 14:171–176

39. Muzaffar S, Jeremy JY, Sparatore A, Del Soldato P, Angelini GD,Shukla N (2008) H2S-donating sildenafil (ACS6) inhibits superoxideformation and gp91phox expression in arterial endothelial cells: roleof protein kinases A and G. Br J Pharmacol 1555:984–994

40. Nagai Y, Tsugane M, Oka J, Kimura H (2004) Hydrogen sulfideinduces waves in strocytes. FASEB J 18:557–559

41. Nashida T, Takuma K, Fukuda S, Kawasaki T, Takahashi T, Baba A,Ago Y, Matsuda T (2011) The specific Na(+)/Ca(2+) exchangeinhibitor SEA0400 prevents nitric oxide-induced cytotoxicity inSH-SY5Y cells. Neurochem Int 59:51–58

42. NishidaM, Sawa T, Kitajima N, Ono K, Inoue H, Ihara H,MotohashiH, Yamamoto M, Suematsu M, Kurose H, van der Vliet A, FreemanBA, Shibata T, Uchida K, Kumagai Y, Akaike T (2012) Hydrogensulfide anion regulates redox signaling via electrophile sulfhydration.Nat Chem Biol 8:714–724

43. Pacak K, Sirova M, Giubellino A, Lencesova L, Csaderova L,Laukova M, Hudecova S, Krizanova O (2012) NF-κB inhibitionsignificantly upregulates the norepinephrine transporter system,causes apoptosis in pheochromocytoma cell lines and prevents me-tastasis in an animal model. Int J Cancer 131:2445–2455

44. Pan L, ZhangX, SongK,WuX, Xu J (2008) Exogenous nitric oxide-induced release of calcium from intracellular IP3 receptor-sensitivestores via S-nitrosylation in respiratory burst-dependent neutrophils.Biochem Biophys Res Commun 377:1320–1325

45. Paul BD, Snyder SH (2012) H2S signalling through proteinsulfhydration and beyond. Nat Rev Mol Cell Biol 13:499–507

46. Prabhakar NR (2012) Hydrogen sulfide (h2s): a physiologic mediatorof carotid body response to hypoxia. Adv Exp Biol 758:109–113

47. Predmore BL, Lefer DJ (2010) Development of hydrogen sulfide-based therapeutics for cardiovascular disease. J Cardiac Transl Res 3:487–498

48. Predmore BL, Lefer DJ, Gojon G (2012) Hydrogen sulfide inbiochemistry and medicine. Antioxid Redox Signal 17:119–140

49. Robinson H, Wray S (2012) A new slow releasing, H2S generatingcompound, GYY4137 relaxes spontaneous and oxytocin-stimulatedcontractions of human and rat pregnant myometrium. PLoS One7(9):e46278. doi:10.1371/journal.pone.0046278

50. Santulli G, Laccarino G (2013) Pinpointing beta adrenergic receptorin ageing pathophysiology: victim or executioner? Evidence fromcrime scenes. Immun Ageing. doi:10.1186/1742-4933-10-10

51. Shao JL, Wan XH, Chen Y, Bi C, Chen HM, Zhong Y, Heng XH,Qian JQ (2011) H2S protects hippocampal neurons from anoxia-reoxygenation through cAMP-mediated PI3K/Akt/p70S6K cell-survival signaling pathways. J Mol Neurosci 43:453–460

52. Sirabella R, Secondo A, Pannaccione A, Scorziello A, Valsecchi V(2009) Anoxia-induced NF-κB-dependent upregulation of NCX1contributes to Ca2+ refilling into endoplasmic reticulum in corticalneurons. Stroke 40:922–929

53. Smith HS (2009) Hydrogen sulfides involvement in modulatingnociception. Pain Physician 12:901–910

54. St Clair JR, Liao Z, Larson ED, Proenza C (2013) PKA-independentactivation of lf by cAMP in mouse sinoatrial myocytes. Channels(Austin) 11:7(4)

55. Staiano RI, Granata F, Secondo A, Petraroli A, Loffredo S,Annunziato L, Triggiani M, Marone G (2013) Human macrophagesandmonocytes express functional Na(+)/Ca (2+) exchangers 1 and 3.Adv Exp Med Biol 961:317–326

56. Tang T, Lai NC, Wright AT, Gao MH, Lee P, Guo T, Tang R,McCulloch AD, Hammond HK (2013) Adenylyl cyclase 6 deletionincreases mortality during sustained β-adrenergic receptor stimula-tion. J Mol Cell Cardiol 60:60–67

57. Tang XQ, Yang CH, Chen J, Yin W, Tian S, Hu B, Feng J, Li Y(2008) Effect of hydrogen sulphide onβ-amyloid-induced damage inPC12 cells. Clin Exp Pharmacol Physiol 35:180–186

58. Tiong CX, Lu M, Bian JS (2010) Protective effect of hydrogensulphide against 6-OHDA-induced cell injury in SH-SY5Y cellsinvolves PKC/PI3K/Akt pathway. Br J Pharmacol 161:467–480

59. Valsecchi V, Pignataro G, Sirabella R, Matrone C, Boscia F,Scorziello A, Sisalli MJ, Esposito E, Zambrano N, Cataldi M, DiRenzo G, Annunziato L (2013) Transcriptional regulation of ncx1gene in the brain. Adv Exp Med Biol 961:137–145

60. WangR (2002) Two’s company, three’s a crowd: can H2S be the thirdendogenous gaseous transmitter? FASEB J 16:1792–1798

61. Wang R (2012) Physiological implications of hydrogen sulphide: awhiff exploration that blossomed. Physiol Rev 92:791–896

62. Yang G, Cao K, Wu L, Wang R (2004) Cystathionine gamma-lyaseoverexpression inhibits cell proliferation via a H2S-dependent mod-ulation of ERK1/2 phosphorylation and p21Cip/WAK-1. J Biol Chem279:49199–49205

63. Yang HY,Wu ZY,WoodM,WhitemanM, Bian JS (2013) Hydrogensulfide attenuates opioid dependence by suppression of adenylatecyclase/cAMP pathway. Antioxid Redox Signal. doi:10.1089/ars.2012.5119

64. Yin WL, He JQ, Hu B, Jiang ZS, Tang XQ (2009) Hydrogen sulfideinhibits MPP(+)-induced apoptosis in PC12 cells. Life Sci 85:269–275

65. Yong QC, Pan TT, Hu LF, Bian JS (2008) Negative regulation ofbeta-adrenergic function by hydrogen sulphide in the rat hearts. JMoll Cell Cardiol 44(4):701–710

66. Zhang JH, Dong Z, Chu I (2010) Hydrogen sulphide induces apo-ptosis in human periodontium cells. J Periodontal Res 45:71–78

67. Zhang LM, Jiang CX, Liu DW (2009) Hydrogen sulfide attenuatesneuronal injury induced by vascular dementia via inhibiting apopto-sis in rats. Neurochem Res 34:1984–1992

68. ZhaoW, Zhang J,Wang R (2001) The vasorelaxant effect of H(2)S asa novel endogenous gaseous K (ATP) channel opener. EMBO J 20:6008–6016

Pflugers Arch - Eur J Physiol